L0301P40 - Gene and Protein Engineering in Clinical Medicine
Biotechnology *the ability to clone genes has made possible many new applications of biotechnology, such as the large-scale production of gene products from a variety of organisms Production of Proteins for Treatment #isolation of gene (cDNA) #introduction of the gene into a host cell system where it is expressed #growth of host cells on a commercial scale #purification of product to meet rigorous standards #extensive testing to establish clinical efficacy #approval from regulatory authorities for clinical use   Expression Vector *turning cells into protein factories *cloned gene is introduced into a vector to facilitate high level expression *host cells include: **bacteria, yeast ***easier to grow **cultured insect or mammalian cells ***better for expression of larger secretory proteins *for a vector carrying a gene of interest to be expressed in a host cell, the gene: **must be adjacent to appropriate sequences for its transcription and translation ***downstream of the promoter ***upstream of the termination sequence **sequences can be added to achieve secretion or facilitate product purification **tags can be added to the gene so that the protein will also express the tag and allow for easier identification at the end Medically Useful Proteins *made by recombinant DNA and expression vectors *are generally otherwise difficult to obtain in sufficient quantities Examples Tissue Plasminogen Activator *TPA produced by cells lining blood vessels *enzyme normally converts plasminogen into plasmin - a protein that dissolves blood clots *helped those who suffered blood clotting in heart attacks or strokes *previously:   **only drug available (Streptokinase - bacterial enzyme) would cause immune reactions if used repeatedly on a patient *now: **produced in E.Coli by recombinant DNA **mRNA has been sourced —> reverse transcriptase to obtain the cDNA which is inserted into E.Coli Human Growth Hormone *synthesised and secreted by the pituitary at the base of the brain *is species specific i.e. only the human protein works in humans *regulates growth and development *used to restore growth in children with hypopituitary dwarfism (HGH deficiency) **must treat early while bone growth is still possible *previously: **expensive: 1 year supply derived from 80 cadavers; need to administer for 8-10 yrs **mid 1980s loss of supply from human organ source: Creutzfeldt-Jacob infection *now: **made in bacteria or mammalian cells Therapies against Infectious Diseases Hepatitis B *no cure available *no vaccine could be produced by standard methods of using inactivated or weakened *virus as they could not be grown in mammalian cell cultures Available Vaccine *antibodies from the serum of people recovered from infection **passively vaccinate acutely infected patients **protection is short-lived lasting only about three months Alternative Strategy *use protein from the virus as an antigen to stimulate the immune response *major surface antigenic protein inducing antibody formation identified **HBSAg (hepatitis B surface antigen) *found in the plasma of infected individuals *successful synthesis in yeast but not in bacteria *tested for effectiveness in primates and then in human volunteers. *released under license in Australia: **EngerixTM -B - claimed 94.5% efficacy *only effective if vaccination takes place before infection Plant Biotechnology *introduce new, useful genes into plants via vectors targeting improvement in nutrition and suitability to environment *previously: conventional breeding *now: biotechnology can insert desired genes for the preferred characteristics Improved Nutritional Characteristics *rice does not have β-carotene, but has the precursor molecule *genes for enzymes that synthesise β-carotene from the precursor are taken from daffodils and inserted into rice *the transgenic rice is yellow, and can supply β- carotene (converted to vitamin A in the body) to improve the diet Concerns *genetic manipulation is an unnatural *genetically altered foods are unsafe to eat *altered crop plants are dangerous to the environment - escape of transgenes into wild populations and create resistance Oral Plant Vaccines *aim is to create plants which can be ingested and used as vaccines *E.g: “hepatitis tomato” - fruit will express viral proteins to act as antigens   Pharming *using animals as bioreactors for producing proteins *E.g.: using transgenic dairy animals to produce useful proteins in their milk DNA Vaccines *directly injected or fired (DNA coated gold beads) into muscle *are based on use of plasmids containing protein coding genes *advantages: **not infectious or replicative **more stable and cheaper than “traditional” vaccines Establishment *cut pathogen DNA into fragments and clone into a plasmid to make a library *divide the library into pools *test pools in animals; give time to develop response then inject pathogen *assume any unaffected animal group contains at least one “protective” plasmid from a pool *subdivide this pool and repeat the testing cycle until a single/few plasmids are identified that confer protection